Interaction between the herpes simplex virus type 1 origin-binding and DNA polymerase accessory proteins.
Interactions between the herpes simplex virus type 1 (HSV-1) origin (ori)-binding protein (UL9) and two other components of the functional DNA replication complex have been observed. However, to date, no interaction between UL9 and a component of the DNA polymerase holoenzyme has been demonstrated. In this report, we demonstrate that UL9 and the DNA polymerase accessory protein (UL42) can form a stable complex in vitro as determined by coimmunoprecipitation with specific antibodies to each protein and by affinity chromatography using glutathione S-transferase (GST) fusion proteins. Complex formation does not require the presence of other viral proteins and occurs in the presence of ethidium bromide, indicating that UL9-UL42 interaction is DNA independent. Affinity beads charged with increasing concentrations of GST-42 fusion protein up to 5 microM bound increasing amounts of UL9 expressed by in vitro transcription/translation in rabbit reticulocyte lysates. Binding of N- and C-terminal portions of UL9 to GST affinity matrices revealed that the N-terminal 533 amino acids were sufficient for binding to GST-42, albeit at approximately a four- to six-fold reduced affinity compared to the full-length protein. No binding of a polypeptide containing the remainder of the UL9 C-terminal residues was observed. Thus the ori-binding protein, UL9, can physically associate with at least one member of each of the complexes (helicase/primase, DNA polymerase holoenzyme, single-stranded DNA-binding protein) required for origin-dependent DNA replication. These specific interactions provide a means by which the ordered assembly of HSV-1 DNA replication proteins at origins of replication can occur in the infected cell for initiation of viral DNA synthesis.
Monahan
S J
SJ
Department of Medical Microbiology and Immunology, Ohio State University, 333 West Tenth Avenue, Columbus, Ohio, 43210, USA.
Grinstead
L A
LA
Olivieri
W
W
Parris
D S
DS
D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000378:metabolism*; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism; D011233:Precipitin Tests; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D014764:Viral Proteins / Q000276:immunology / Q000378:metabolism*
D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C064565:UL9 protein, Human herpesvirus 1; D005982:Glutathione Transferase; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus